雷帕霉素介导的caspase 9同源二聚化调控人类多能干细胞自杀
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国家自然科学基金(82001974);广东益阳医疗慈善基金(JZ2022018)


Rapamycin mediated caspase 9 homodimerization to safeguard human pluripotent stem cell therapy
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    摘要:

    人诱导多能干细胞(human induced pluripotent stem cells,hiPSCs)在再生医学领域具有广阔的应用前景。然而,多能干细胞(pluripotent stem cells,PSCs)具有肿瘤化风险,成为其临床应用最主要的安全性问题。雷帕霉素是一种安全和广泛使用的免疫抑制药物,通过诱导FKBP12与FRB片段的异源二聚起作用。为了保障hiPSCs治疗的安全性,本研究将雷帕霉素诱导的caspase 9(riC9)基因插入到AAVS1安全位点,构建了含有EF1α启动子、FRB-FKBP-Caspase9(CARD结构域)融合蛋白和嘌呤霉素抗性基因的供体,并与sgRNA/Cas9载体共转染hiPSCs。用嘌呤霉素筛选2周后,收集单个克隆进行基因和表型分析。最后,用雷帕霉素诱导caspase9同源二聚化,激活工程细胞的凋亡。通过对筛选获得的5个hiPSCs克隆鉴定,表明供体DNA准确敲入内源性AAVS1位点。hiPSCs保持正常的多潜能状态和增殖能力。雷帕霉素可诱导caspase 9同源二聚化,并激活细胞凋亡程序。本研究通过药物精确调控caspase 9的同源二聚化来启动细胞自杀,实现了可控的hiPSCs存活,这为保证hiPSCs治疗的安全性提供了新的策略。

    Abstract:

    Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue, which is a major safety concern in PSCs therapies. Rapamycin is a safe and widely used immunosuppressive pharmaceutical that acts through heterodimerization of the FKBP12 and FRB fragment. Here, we aimed to insert a rapamycin inducible caspase 9 (riC9) gene in a safe harbor AAVS1 site to safeguard hiPSCs therapy by drug induced homodimerization. The donor vector containing an EF1α promoter, a FRB-FKBP-Caspase 9 (CARD domain) fusion protein and a puromycin resistant gene was constructed and co-transfected with sgRNA/Cas9 vector into hiPSCs. After one to two weeks screening with puromycin, single clones were collected for genotype and phenotype analysis. Finally, rapamycin was used to induce the homodimerization of caspase 9 to activate the apoptosis of the engineered cells. After transfection of hiPSCs followed by puromycin screening, five cell clones were collected. Genome amplification and sequencing showed that the donor DNA has been precisely knocked out at the endogenous AAVS1 site. The engineered hiPSCs showed normal pluripotency and proliferative capacity. Rapamycin induced caspase 9 activation, which led to the apoptosis of all engineered hiPSCs and its differentiated cells with different sensitivity to drugs. In conclusion, we generated a rapamycin-controllable hiPSCs survival by homodimerization of caspase 9 to turn on cell apoptosis. It provides a new strategy to guarantee the safety of the hiPSCs therapy.

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杨洋,刘洋,陈敏,李双鹏,鹿璇,贺瑜,张焜,邹庆剑. 雷帕霉素介导的caspase 9同源二聚化调控人类多能干细胞自杀[J]. 生物工程学报, 2023, 39(10): 4098-4107

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  • 收稿日期:2023-02-09
  • 录用日期:2023-04-06
  • 在线发布日期: 2023-10-17
  • 出版日期: 2023-10-25
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